Figure 7

Internalized Pg381 and MFI microorganism initiation or undergoing division and intracellular survival in ARPE-19 cells. (A–A1) SEM showed evidence of Pg undergoing division on the surface of infected ARPEs at 10 MOI for 1 hour. (B–D) TEM images illustrate the internalized Pg381 and MFI (10 MOI) microorganism initiation (dotted blue arrow) or undergoing (dotted red arrows) division and survival in ARPE for 1 (B, B1) and 24 (C-C1 & D-D1) hours is evident. We observed the apparent contact between micro-filamentous cellular components (black arrows) and surface-adhering P. gingivalis (red arrows) while undergoing division (B-B1) at 1 hour. Evidently, several of the TEM images show, Pg381 (C-C1) and MFI (D-D1) dividing within the ARPE cells at 24 hours. This indicates that the bacteria are metabolically active during invasion and may be able to persist in the ARPE cells for at least 24 hours. Note- Massive extracellular/intracellular bacteria, especially in the case of Pg381 and MFI, appear to be aggregated at certain spots along the cellular membrane and cytosol. Also refer the second set of low to high magnified TEM images in Fig. S7 for the complete view and confirmation. A1, B1, C1 and D1 shows a magnified/enlarged region of Boxed areas in A, B, C and D, respectively. (Scale bar:- A-D: 2 µm, B: 1 µm, B1: 0.5 µm and A1, C1, D1: enlarged from A, C and D, respectively). (E) The figure shows the replication of Pg-strains at 1-hour infection and 5 hours post invasion; the cells were lysed, Pg381, MFI and DPG3 recovered and CFU enumerated as described in the methods. The data represents CFU within ARPE cells harvested from biological triplicates. The mean ± SD were analyzed by One-way ANOVA of different groups and Tukey’s test for multiple group comparisons within 3 different experiments with 3 technical replicates from each experiments. (n = 9; ***P < 0.001). CFU- Colony Forming Unit. Refer Fig. S9 for Pg strains replication/colony formation.